Recombinant protein expression involves using living cells to produce a specific protein based on genetic instructions. This technique is widely used in scientific research to study protein function and structure. Beyond research, it holds importance in medicine, enabling the production of therapeutic proteins like insulin, and in various industrial applications.
Bacteria, particularly Escherichia coli (E. coli), serve as a preferred host system for this process. Their rapid growth, low cost of cultivation, and well-understood genetic makeup make them advantageous. Over decades, scientists have genetically engineered E. coli strains to optimize them for efficient and high-yield protein production. This article explores common genetically engineered E. coli strains for recombinant protein expression and guides selection for specific needs.
The BL21 Strain Family
The BL21 strain of E. coli is a common choice for recombinant protein expression. This particular strain is derived from the B lineage of E. coli, which naturally exhibits a deficiency in the Lon protease. This deficiency reduces the degradation of newly synthesized proteins.
Further genetic modifications enhance the utility of BL21 and its derivatives for expressing foreign proteins. One significant alteration involves the deletion of both the lon and ompT protease genes. The absence of these major intracellular proteases minimizes the breakdown of the target recombinant protein, leading to higher yields. This preserves the protein’s integrity and quantity.
The T7 expression system is central in many BL21 derivatives, most notably BL21(DE3). In this system, the gene for T7 RNA polymerase is integrated directly into the E. coli chromosome. This integration places the T7 RNA polymerase gene under the control of the lacUV5 promoter, which allows for regulated protein synthesis.
Adding isopropyl β-D-1-thiogalactopyranoside (IPTG) induces this system. IPTG binds to the Lac repressor, releasing its inhibition on the lacUV5 promoter. This permits transcription of the T7 RNA polymerase gene, producing T7 RNA polymerase. The T7 RNA polymerase then transcribes the target gene located on a high-copy number expression plasmid, such as a pET vector, at a high rate. This regulated induction ensures that the protein is produced only when desired, preventing toxicity or metabolic burden.
Overcoming Codon Bias
Differences in codon usage among organisms challenge recombinant protein expression. Codon usage bias occurs when different organisms prefer specific synonymous codons (DNA triplets that code for the same amino acid). For instance, a gene originating from a human or plant might utilize codons rarely employed in E. coli.
When E. coli encounters these rare codons, it may lack sufficient corresponding transfer RNA (tRNA) molecules. This scarcity can lead to problems during protein synthesis. Translation can stall, leading to premature termination and the production of truncated proteins. This reduces the yield of full-length target protein.
To address this issue, specialized E. coli strains have been developed, such as those in the Rosetta™ series. These strains are often derivatives of BL21(DE3) and carry an additional plasmid. This plasmid contains genes that encode tRNAs recognizing codons rare in E. coli but common in many eukaryotic organisms.
These supplemented tRNAs include those for codons like AGG, AGA (arginine), AUA (isoleucine), and CUA (leucine). By supplying these rare tRNAs, these strains facilitate more efficient translation of heterologous genes. This prevents translational stalling and promotes the production of full-length, soluble recombinant proteins. Other strains, such as the CodonPlus series, also solve codon bias by supplying the necessary tRNA genes.
Solutions for Challenging Proteins
Some recombinant proteins are difficult to express in standard E. coli strains due to their biochemical characteristics. Proteins requiring disulfide bonds for proper folding and function often face challenges. The cytoplasm of typical E. coli strains maintains a chemically reducing environment.
This reducing environment prevents the formation of stable disulfide bonds, covalent linkages between cysteine residues often necessary for the correct three-dimensional structure of many eukaryotic proteins. Without these bonds, the target protein may misfold, aggregate, or become inactive. Strains like SHuffle® Express address this problem. These strains possess a modified, oxidizing cytoplasm that supports disulfide bond formation. This environment is created through specific genetic mutations (e.g., in the gor and trxB genes) that disrupt the reductive pathways.
Another challenge is when the recombinant protein itself is toxic to the E. coli host. Even minimal “leaky” expression from the T7 promoter before induction can lead to cell death or inhibit bacterial growth. This basal level of expression can be detrimental, especially for proteins that interfere with fundamental cellular processes.
Specialized strains like C41(DE3) and C43(DE3) mitigate this toxicity. These strains carry mutations that reduce T7 RNA polymerase activity, leading to lower, more manageable expression levels less toxic to the host cells. Strains containing plasmids such as pLysS or pLysE offer another layer of control. These plasmids produce T7 lysozyme, which inhibits basal T7 RNA polymerase activity, ensuring tighter regulation and minimizing leaky expression of toxic proteins.
Choosing the Right Expression Strain
Selecting the appropriate E. coli strain for recombinant protein expression optimizes yield and solubility. Begin with a standard, robust strain, especially when working with a new or uncharacterized protein. BL21(DE3) is a reliable starting point for many expression projects.
After an initial attempt, evaluate the results and consider specific modifications to the expression system. If the protein is from a distantly related organism (e.g., human or plant) and initial yields are low, codon bias may be a factor. In such cases, switching to a codon-optimized strain like Rosetta 2(DE3) can improve translational efficiency by supplying tRNAs for rare codons.
For proteins requiring disulfide bonds for structural integrity and biological activity, a standard E. coli cytoplasm is unsuitable. If disulfide bond formation is needed, use a strain with an oxidizing cytoplasm, such as SHuffle Express. These strains facilitate the correct folding of disulfide-bonded proteins in the bacterial cytoplasm.
Finally, if the recombinant protein is toxic to host cells (indicated by poor bacterial growth before induction), a different strategy is needed. Using a strain designed for tighter expression control or lower expression levels (e.g., a pLysS host or C41(DE3)) can alleviate the cellular burden. These specialized strains help maintain cell viability while still allowing for controlled protein production.